Protein Phosphorylation And regulation of Cytoskeleton in Nervous system In the nervous system: The cytoskeletal proteins are extensively phosphorylated in the nervous system. Most of this phosphorylation occurs on the proline-directed serine/threonine (S/TP) residues of cytoskeletal proteins, such as neurofilament high and medium molecular weight (NF-H and NF-M) and the microtubule-associated proteins MAP-2 and tau. Normally, these proteins are phosphorylated selectively in the axonal compartment of the neuron. Although all kinases, phosphatases, their substrates and their regulators are synthesized in the cell body, little or no cytoskeletal protein phosphorylation on S/T residues is detected in the cell body. This compartmentalization of phosphorylation is tightly regulated. However, in a number of neuropathological conditions, such as amyotrophic lateral sclerosis (ALS), Alzheimer?s disease (AD) and Picks Disease it gets deregulated. There is an aberrant phosphorylation of these cytoskeletal proteins on S/T residues, in the cell bodies where they accumulate in aggregated form. This leads to massive neuronal cell death in these affected areas. The major focus has been to identify the kinases that phosphorylate these S/T residues. In search of these kinases, we discovered that in addition to the mitogen activated protein kinases (MAPKs) e.g. ERK1/2, Cdk5 is another major kinase that phosphorylates multiple lysine/serine/proline (KSP) repeats in the carboxy-terminal domains of NF-M, NF-H, and tau. Cdk5 was purified from brain tissue and also was cloned from a rat cDNA library in our laboratory. We have demonstrated that various signal transduction cascades in primary neuronal cultures and transfected non-neuronal cells activate cdk5. Myelinating glial cells are a major source of exogenous activating signals. Using myelin associated glycoprotein (MAG) knockout mice, which exhibit no myelination, we have shown that ERK1/2 and Cdk5 activities in brain and sciatic nerve extracts are down-regulated as compared to wild-type mice. These studies also indicated that increased ERK1/2 and Cdk5 activity induced by glial-axon interactions stimulated axonal phosphorylation of NF-M, NF-H, MAP-2B and tau. We have concluded that MAG may act as a ligand to activate the appropriate kinase cascade causing axonal cytoskeletal protein phosphorylation. In addition, matrix elements e.g. integrins may also activate these kinases . More recently, our laboratory has focused to study the role of cdk5 in nervous system function and development. Although Cdk5 is ubiquitously expressed in all cells and shares a high degree of homology with other members of the cyclin-dependent kinase family (Cdks), its activity is found specifically in post-mitotic neurons because its activators, p35 and p39 are expressed primarily in neurons. Studies from different laboratories, including ours, have shown that Cdk5 is a multi-functional S/T protein kinase that is involved in a wide range of neuronal functions from neurite outgrowth and neuronal migration to synaptic activity and cell survival. We have shown, for example, that cdk5 KO mice (-/-) are lethal, exhibiting abnormal corticogenesis and other neuronal abnormalities before dying between E16 and P0. Recently, we have also demonstrated that experimental re-expression of cdk5 in neurons of cdk5 KO mice in vivo completely restored the wild type, clearly demonstrating that neuronal and not glial cdk5 activity is necessary for normal development and survival . The diverse roles of cdk5 are based, in part, on evidence that it is a key player in signal transduction networks underlying neuronal cell survival, growth and differentiation. We observed the presence of hyperphosphorylated cytoskeletal proteins in swollen brain stem and spinal cord perikarya in cdk5-/- mice. This led us to look for other kinases affected by the absence of cdk5. Since cdk5 activity is down regulated in p35-/-mice, we found that MAPK (Erk1/2) was hyper-activated in brain extracts which further suggested that cdk5 modulated a site in the MAP kinase cascade.These results suggest that Cdk5 is involved in "cross-talk" with other signal transduction and survival pathways, perhaps acting to modulate the intensity of the response to specific signals. . Cdk5 activity is tightly regulated in the nervous system and it may promote neuronal cell survival or induce programmed cell death (PCD). The response depends on cell type, its state of proliferation or differentiation, the nature of exogenous signals, and the specific signaling pathways involved. The cdk5 (-/-) lethal phenotype suggests that cdk5 may be involved in the survival pathway since elevated levels of apoptosis were seen in cortical brain regions of E16-18 embryos. To explore this further, we studied the effect of cdk5 on the well-studied JNk3-mediated apoptotic pathway. We could show that the apoptotic effects of the activated c-Jun N-terminal kinase 3 (JNk3) can be inhibited by cdk5/p35, another illustration of cdk5 cross talk with signal transduction pathways, in this case, with one involved in PCD. We demonstrated that cdk5/p35 could phosphorylate JNk-3 on Thr131 and down regulate its activity and phosphorylation of c-jun. Moreover, in vivo studies showed that Jnk3 activity was elevated in brain extracts of cdk5 KO mice compared to wild type, which could explain the increased cortical apoptosis. UV-induced apoptosis was also increased in cdk5 -/- cortical neurons in culture which exhibited elevated levels of caspase expression, consistent with a role for cdk5/p35 in protecting against a Jnk-3 activated apoptotic cascade. An additional clue about cdk5's role in apoptosis came from the cdk5 KO mice. We observed that the level of phosphorylated phospholipid is considerably reduced in Cdk5 knockout brain, suggesting that phospholipid inositol-3 kinase (PI3-kinase) activity is compromised. PI3K is a key kinase, which phosphorylates Akt kinase downstream in a survival-signaling cascade suggesting a role for cdk5 in neuronal survival during development. It has been proposed that deregulation of cdk5 activity in the brain, by abnormal production of p25, a truncated, more active fragment of p35, can lead to hyperphosphorylated tau pathologies characteristic of AD brains in humans . Our study of site specific interactions between cdk5 and truncated forms of its p35 regulator have revealed a central p35 fragment, 125 amino acids residues (CIP) that has high affinity for and inhibits the in vitro activity of the Cdk5/p25 complex. We have shown that CIP specifically inhibits Cdk5/p25 activity in transfected cells and also reduces phosphorylation of co-transfected tau. It is important to note that CIP does not affect the activity of cdc2 kinase. The question, however, arises as to whether CIP will inhibit cdk5/p25 activity in primary neurons. If CIP specifically inhibits cdk5 hyperactivity in primary neurons, can it inhibit the hyperphosphorylation of tau and NF proteins in AD model phenotypes induced in p25 transgenic mice and in an ALS transgenic model?